1. Field Of The Invention
This invention relates to the use of t-PA and a heparin fraction which has low affinity for t-PA in a composition for thrombolytic therapy.
2. Background Of The Related Art
Pathologies of blood coagulation such as heart attacks, strokes, and the like, account for approximately fifty percent (50%) of all hospital deaths. These diseases are caused by the development of thromboses, or blood clots, at inappropriate locations within the cardiovascular system. Blood clot formation derives from a series of events, called the thrombolytic cascade, in which the final steps involve the formation of the enzyme thrombin. Thrombin converts circulating fibrinogen protein into fibrin, a mesh-like structure which forms the blood clot. The thrombolytic cascade is highly regulated, it can be suppressed by heparin, which inhibits the coagulation of blood. See FIG. 1, Scheme I. shows the Thrombolytic Cascade, the clot formation process in which thrombin joins with circulating fibrinogen in the blood stream to form fibrin, the mesh-like structure which makes up the clot. Scheme II shows the fibrinolytic, i.e. the clot dissolution mechanism of the Thrombolytic Cascade.
The life-saving process of clot production in response to an injury can become life-threatening when it occurs at inappropriate places in the body. For example, a clot can obstruct a blood vessel and stop the supply of blood to an organ or other body part. Equally life-threatening are clots that become detached from their original sites and flow through the circulatory system causing blockages at remote sites, these clots are called embolisms.
The Thrombolytic Cascade involves a series of transformations due to a succession of the zymogen activations. In the Thrombolytic Cascade, the activated form of one enzyme factor initiates the activation of the next enzyme. The numerous steps of the Cascade yield a large amplification factor to assure a rapid response to trauma or injury. Clotting, therefore, involves the interplay of two systems: the intrinsic pathway which can be triggered by contact with charged surfaces, e.g. glass, and the extrinsic pathway which is triggered by trauma to the tissue. These two systems converge to a final common pathway that results in the production of the fibrin clot, a transformation mediated by thrombin, a proteolytic enzyme. Thrombin is responsible for the conversion of the highly soluble fibrinogen, into insoluble fibrin.
The conversion of fibrinogen to fibrin can be inhibited by a plasma protease inhibitor called Antithrombin III ("AT III"). AT-III is a relatively slow inhibitor of thrombin, although in the presence of heparin, the rate of inhibition of thrombin by AT-III is considerably enhanced Thus, heparin is a very powerful blood clotting inhibitor.
The clot, however, once formed, can eventually be dissolved by a group of enzymes of the fibrinolytic system. A major component of the fibrinolytic system is a plasma protein called plasminogen which can be converted in the blood to plasmin. Formation of plasmin is mediated by other enzymes called plasminogen activators.
Plasminogen activators ("PA's") are serine proteases which convert the proenzyme plasminogen into plasmin, an enzyme with a broad substrate specificity. When circulating freely in the blood, plasmin degrades several proteins, including fibrinogen and some coagulation factors. Two distinct types of PA's are known; urokinase plasminogen activator ("u-PA") and tissue-type plasminogen activator ("t-PA"). Both u-PA and t-PA are currently being used as thrombolytic agents, see for example, Verstraete, et al., Blood, 67, 1529 (1986); t-PA is widely preferred over u-PA, since t-PA's activity is strongly stimulated by the presence of fibrin, the main component of blood clots. See, Hoylaerts et al., J. Biol. Chem., 257, 2912 (1982). Thus, intravenously injected t-PA could induce a limited systematic lysis of fibrinogen and other substrates with the majority of its plasmin-generating activity confined to the areas of fibrin clots.
Four major clinical trials have examined the effectiveness of t-PA for thrombolysis in myocardial infarction; see, Collen et al., Circulation, 70, 1012 (1984); The TIMI Study Group, New England Journal of Medicine, 312, 932 (1985); Verstraete M. et al., Lancet, 1, 842 (1985); and Verstraete, M. et al., Lancet, 2, 965 (1985). In these trials, streptokinase, a non-enzyme protein produced by strains of .beta.-hemolytic streptocci, was also tested for its properties as an indirect activator of the fibrinolytic system. These trials have shown that the frequency of bleeding complications was not significantly different for all agents tested. Also, systemic fibrinogenolysis was still apparent in t-PA treated patients, although it was less pronounced than with either streptokinase or u-PA.
Heparin also interacts with the fibrinolytic system. The interaction of heparin with t-PA in the fibrinolytic system results in an increase in the stimulation by t-PA of the conversion of plasminogen to plasmin. In addition, heparin binds to t-PA and decreases the stimulatory effect of fibrin on the action of t-PA.
The ability to specifically dissolve abnormal blood clots has long been a profoundly important clinical goal. The study of the intricate system of physiological thrombolysis and fibrinolysis (the dissolution of fibrin) has been a rapidly growing field which has resulted in the development of a new generation of thrombolytic agents.
Previous therapeutic treatments for dissolving life-threatening clots have included injecting into the blood system various enzymes which are known to break down fibrin. The problem with these treatments is that the enzymes were not site specific, and therefore, would do more than just cause dissolution of the clot. In addition, these enzymes interfere with and destroy many vital protein interactions that serve to keep the body from excessively bleeding due to the many minor injuries it receives on a daily basis. Destruction of these safeguards by these enzymes can lead to serious hemorrhaging and other potential fatal complications. See FIG. 1, Scheme II.
Recently, t-PA has shown promise as a thrombolytic agent. Because the activity of t-PA must be stimulated by the presence of fibrin, injected t-PA normally has little activity in the mammalian, such as human, circulatory system. In the vicinity of a clot, however, the presence of fibrin stimulates t-PA activity which then stimulates the conversion of plasminogen to plasmin to degrade the clot.
Even though t-PA occurs naturally in the mammalian (e.g. human) circulatory system, it is difficult to isolate, and until recently, naturally occurring t-PA has only been available for therapeutic use in very small quantities. Recent advances in genetic engineering have made it possible to produce large amounts of recombinant t-PA ("rt-PA") for research and clinical use. See U.S. Pat. Nos. 4,752,603; 4,751,084; and 4,753,879, the entire disclosures of which are incorporated by reference herein The large scale production and use of t-PA alone, however, has not solved all of the problems endemic to clotting disorders. One severe problem with the use of t-PA in clinical trials of human patients has been the rapid reformation of the clot after it has been dissolved. This clot reformation, called reocclusion, usually occurs within minutes of the clot being dissolved
To prevent reocclusion, heparin, which inhibits blood coagulation, has been given in conjunction with t-PA therapy, Heparin is a complex glycosaminoglycan, illustrated in FIG. 2, isolated from a variety of natural sources. Usually, heparin is injected prior to, during, and after treatment with t-PA. Heparin is a valuable anticoagulant which acts catalytically to disrupt the action of thrombin. Previous publications have indicated that heparin can induce fibrinolysis in vivo and in vitro; see for example, Vinazzer et al., Influence of Heparin; of Different Heparin Fractions and of a Low Molecular Weight Heparin-Like Substance on the Mechanisms of Fibrinolysis, Throm. Res., 27, 341 (1982); however, the mechanisms of interaction of heparin in the thrombolytic cascade have remained obscure. The combination of t-PA and heparin seem to be the key to effectively dissolving blood clots and preventing their reocclusion in heart attack victims.
An article by Turpie et al., A Randomized Controlled Trial of a Low-Molecular-Weight Heparin (Enoxaparin) to Prevent Deep-Vein Thrombosis in Patients Elective Hip Surgery, New England Journal of Medicine, 315, 925-929 (1986), attributes the reduced hemorrhagic effects of a low molecular weight heparin fraction to lower inhibition of platelet function by the low molecular weight heparin fraction than by standard heparin, and a higher antithrombotic activity observed in these experiments was attributed to higher levels of the low molecular weight heparin. Turpie et al. conclude it is unlikely that the low molecular weight heparin fraction is inherently more antithrombotic than standard heparin.
The previously mentioned article by Vinazzer, et al., discloses that the magnitude of fibrinolysis depends on the degree of sulfation in heparin fractions since a low molecular weight heparin fraction with a high number of sulfate bonds was considerably more active in fibrinolysis than a low molecular weight fraction of standard heparin. An editorial by Sherry, Tissue Plasminogen Activator (t-PA) Will It Fulfill Its Promise?, in the New England Journal of Medicine, Vol 13, No. 16, 1014-1017 (1985), discusses the problems of bleeding complications resulting from recombinant t-PA therapy in conjunction with simultaneous heparin therapy to prevent rethrombosis, but does not suggest any manner of solving those problems.
An article by deProst, Heparin Fractions and Analogues: A New Therapeutic Possibility For Thrombosis, Trends in Pharmacological Sciences, 7, 496-500 (Dec. 1986), discloses reduced interaction with platelets by low molecular weight heparin fractions and natural or semi-synthetic heparin analogues and the longer duration of action in vivo of these compounds compared to heparin. The article suggests investigation into the mechanisms of action of these fractions and analogues of heparin to define their indications in treatment of thrombosis, but does not disclose how these investigations may be carried out.
Paques et al., Study on the Mechanism of Action of Heparin and Related Substances on the Fibrinolytic System: Relationship Between Plasminogen Activators and Heparin, Thrombosis Research 42, 797-807 (1986) discloses that t-PA and u-PA bind tightly to heparin-SEPHAROSE, and that the plasminogenolytic and the fibrinogenolytic activity of t-PA and u-PA can be stimulated at low unfractionated heparin concentrations.
A human pharmacological study comparing conventional heparin and a low molecular weight heparin fragment was reported by Bratt et al., in Thromb. Haemostasis, 53, 208-211 (1985).
Previous results by the inventors, herein, Andrade-Gordon, P., and Strickland, S., were reported in Interaction of Heparin with Plasminogen Activators and Plasminogen: Effects on the Activation of Plasminogen, Biochemistry, 25, 4033 (1986), and have shown that heparin interacts with t-PA, thus, interfering with t-PA's site specific action.
One problem with the use of heparin with t-PA is that the activity of t-PA which, in addition to being stimulated by fibrin, as previously noted, is also stimulated by heparin. Accordingly, the concomitant injection of heparin and t-PA in the mammalian circulatory system has two potentially deleterious consequences. First, heparin could activate t-PA in the general circulatory system, causing destruction of blood components with resulting hemorrhaging Second, heparin inhibits the binding of t-PA to fibrin and could prevent the localization of the enzyme at the site of the clot. See, FIG. 1, Scheme 2. The cumulative affect of both of these actions would reduce the specific fibrin-(clot)-dependent activity of t-PA and increase the non-specific protein degradation, both of which are therapeutically undesirable.
In their aforementioned article, the inventors herein primarily describe the activation of plasminogen by unfractionated heparin and set forth their theory to explain the interaction between heparin, t-PA and u-PA in plasminogen activation In addition, in that article, two fractions of heparin separated on the basis of their affinity for antithrombin-III-SEPHAROSE differed greatly in their ability to stimulate AT-III activity, although not differing in their enhancement of t-PA mediated plasmin activity.
More recently, the inventors herein have provided an overview of their work in New Strides in Immediate Treatment for Heart Attacks, Biotechnology Network, a newsletter of the State University of New York at Stony Brook (May/June 1987). In this article, the inventors herein speculate that it might be possible to fractionate heparin and separate the components responsible for anticoagulation activity from those components that activate t-PA. The inventors also propose the possibility of using recombinant DNA techniques to produce a t-PA enzyme which may be effective in clot dissolution but would not interact with heparin. No suggestion is made, however, of how to achieve these goals. Nor is there any discussion of using these modified components in combination therapy.
The aforementioned articles explore the action of heparin, its various components and analogues, but none describes a heparin fraction which can be safely used in conjunction with t-PA to prevent reocclusion without unwanted stimulation of, or interference with t-PA activity.
Accordingly, it is an object of the present invention to provide an effective thrombolytic therapy for treating mammals, including humans without reducing specific fibrin- o dependent activity and without increasing non-specific protein degradation.
It is a further object of this invention to provide a heparin fraction and a method for its isolation which, in conjunction with t-PA therapy in mammalian, including human patients is effective in preventing reocclusion without causing uncontrolled bleeding by stimulating or interfering with t-PA activity.
It is also an object of the present invention to provide a therapeutic composition which is site specific.
Another object of the present invention is to provide a therapeutic composition which prevents reocclusion of the clot.
It is yet another object of the present invention to provide a new composition for thrombolytic therapy which does not inhibit the binding of t-PA to fibrin, so that t-PA can localize at the site of the clot.
Still another object of the present invention is to provide a new composition for thrombolytic therapy which can inhibit thrombosis without accelerating t-PA activity in the general circulation causing destruction of blood components and resulting in hemorrhage.
Furthermore, it is an object of the present invention to provide a method of thrombolytic therapy with which reocclusion does not occur and without interference with t-PA and fibrinogen function.